HEK293 Cell Culture and Transfection

HEK 293 cells were originally obtained from ATCC and cultured and transfected essentially as previously described (15 (link), 16 (link), 17 (link)). The cells were maintained in DMEM containing 10% fetal bovine serum (both Life Technologies), incubated at 37 °C in 5% CO₂, and used between passage 18 and 30. HEK293 cells used in this study do not express endogenous BK channel subunits as determined by mRNA, protein, or functional assays (15 (link), 16 (link), 17 (link)). For transfection, the cells were typically plated on 6-well plates for 24 h before transfection with corresponding plasmid cDNA using PolyJet (tebu-bio). In all co-transfection assays, channel cDNAs were transfected at a 1:1 ratio with acyl thioesterases or empty pcDNA3.1 plasmid in controls.